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Asz1 is essential for germ cell survival during early gonad development. A. Wt and asz1-/- gonads at 4 wpf as shown on Figs 1 and 2. At 4 hpf, prior to sex determination, all gonads are still developing as ovaries as shown in wt and asz1+/-. asz1-/- exhibit much smaller thread like gonads (white arrows in F) already at 4hpf. Yellow arrows in F indicate surrounding bright fat tissue. B. Distribution of gonad phenotypes as shown in A. n = number of gonads. C-F. Confocal images of wt (C-D) and asz1-/- (E-F) gonads, labeled for mAb414 and Ddx4, as well as β-Catenin and cCaspase3, as shown as in Figs 1 and 2. Developing ovaries in wt contain oogonia and early differentiating oocytes (Ddx4, green) with normally organized perinuclear granules (mAb414, red), but much fewer germ cells in asz1-/- that also exhibit coalesced mAb414 signals (white arrowheads in I). n = 6 gonads per genotype. cCaspase3 labeling (green) of wt, asz1+/-, and asz1-/- gonads. Cells with coalesced mAb414 signals in asz1-/- gonads (F), which are likely abnormal germ cells, are positive for cCaspase3 (white arrowheads in F). n = 5 gonads per genotype. Scale bars in are 50 μm and 10 μm in zoomed out and inset magnification images, respectively. G-H. asz1+/+;Tg(ddx4:GFP) and asz1-/-;Tg(ddx4:GFP) gonads labeled for GFP (magenta), cCspas3 (cyan), and DAPI (blue), showing germ cell apoptosis. Scale bars are 15 μm. I. The number of Ddx4-positive germ cells per gonad is plotted for each genotype. n = number of gonads. Bars are mean ± SD. J. The number of cCaspase3-positive cells are per gonad is plotted for each genotype. n = number of gonads. Bars are mean ± SD. K. % of Ddx4+ germ cells that are cCaspas3+ is plotted. n = number of gonads. Bars are mean ± SD.
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